and cytotoxic effect of ibuprofen and
acetaminophen in an epithelial ovarian
cancer cell line
in vitro
Perfil da expressão gênica dos transportadores ABC e efeito citotóxico
do ibuprofeno e acetaminofen em uma linhagem celular de câncer
epitelial de ovário in vitro
FláviA PeRRimde melo JosiAne BARBosA PiedAde3 PAulA vieiRA teixeiRA vidigAl4 luCiAnA mARiA silvA3 AgnAldo loPesdA silvA Filho2
Abstract
PURPOSES: To determine the basic expression of ABC transporters in an epithelial ovarian cancer cell line, and to investigate whether low concentrations of acetaminophen and ibuprofen inhibited the growth of this cell line in vitro. METHODS: TOV-21 G cells were exposed to different concentrations of acetaminophen (1.5 to 15 μg/mL) and ibuprofen (2.0 to 20 μg/mL) for 24 to 48 hours. The cellular growth was assessed using a cell viability assay. Cellular morphology was determined by luorescence microscopy. The gene expression proile of ABC transporters was determined by assessing a panel including 42 genes of the ABC transporter superfamily. RESULTS: We observed a signiicant decrease in TOV-21 G cell growth after exposure to 15 μg/mL of acetaminophen for 24 (p=0.02) and 48 hours (p=0.01), or to 20 μg/mL of ibuprofen for 48 hours (p=0.04). Assessing the morphology of TOV-21 G cells did not reveal evidence of extensive apoptosis. TOV-21 G cells had a reduced expression of the genes ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 within the ABC transporter superfamily. CONCLUSIONS: This study provides in vitro evidence of inhibitory effects of growth in therapeutic concentrations of acetaminophen and ibuprofen on TOV-21 G cells. Additionally, TOV-21 G cells presented a reduced expression of the ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 transporters.
Resumo
OBJETIVOS: Determinar a expressão básica dos transportadores ABC em uma linhagem celular do câncer epitelial de ovário, e investigar se o acetaminofen e o ibuprofeno em baixas concentrações são capazes de inibir o crescimento desta linhagem celular in vitro.MÉTODOS: A linhagem celular TOV-21 G foi exposta a diferentes concentrações
de acetaminofen (1,5 a 15 µg/mL) e ibuprofeno (2,0 a 20 µg/mL), de 24 a 48 horas. O crescimento celular foi avaliado utilizando-se um ensaio de viabilidade celular. A morfologia celular foi determinada por meio da microscopia de luorescência. O peril de expressão gênica foi estabelecido por um painel de 42 genes da superfamília de transportadores ABC. RESULTADOS: Observou-se um decréscimo signiicativo no crescimento das células TOV-21 G expostas a 15 µg/mL de acetaminofen durante 24 (p=0,02) e 48 horas (p=0,01), ou a 20 µg/mL de ibuprofeno por 48 horas (p=0,04). Ao avaliar a morfologia das células cultivadas, não foi observada evidência de apoptose extensiva. A linhagem de células estudada subexpressa os genes de ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 e ABCE1 na superfamília de transportadores ABC. CONCLUSÕES: Este estudo fornece evidências in vitro referentes aos
efeitos inibidores do crescimento de concentrações terapêuticas do acetaminofen e ibuprofeno na linhagem celular testada. Além disso, as células TOV-21 G apresentaram uma expressão reduzida de genes dos transportadores ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 e ABCE1.
Department of Obstetrics and Gynecology, School of Medicine, Universidade Federal de Minas Gerais – UFMG – Belo Horizonte (MG), Brazil. 1Hospital das Clínicas de Minas Gerais – Belo Horizonte (MG), Brazil.
2Department of Obstetrics and Gynecology, Universidade Federal de Minas Gerais – UFMG – Belo Horizonte (MG), Brazil. 3Laboratory of Research and Development Management, Ezequiel Dias Foundation – Belo Horizonte (MG), Brazil. 4Department of Pathology, Universidade Federal de Minas Gerais – UFMG – Belo Horizonte (MG), Brazil.
Conlict of interests: none. Keywords
Acetaminophen Apoptosis ATP-binding cassette transporters Cell proliferation Chemoprevention Drug therapy Ibuprofen Ovarian neoplasms
Palavras-chave
Acetaminofen Apoptose
Transportadores de cassetes de ligação de ATP
Proliferação de células Quimioprevenção Quimioterapia Ibuprofeno Neoplasias ovarianas
Correspondence Agnaldo Lopes da Silva Filho Department of Gynecology and Obstetrics of Medicine School of Universidade Federal de Minas Gerais Avenida Professor Alfredo Balena, 190 – Santa Eigênia Zip code: 30130-100 Belo Horizonte (MG), Brazil
Received 02/03/2015
Accepted with modiications 05/18/2015
Introduction
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy1,2. Given the poor prognosis for women with EOC, it is imperative to continue exploring
novel chemo-preventive and chemotherapeutic agents3.
A range of indings, from epidemiological studies of patients to molecular studies of genetically modiied mice, has led to a general acceptance that inlammation and cancer are linked4,5. Research focusing on colorectal and prostate cancers has provided strong evidence that nonsteroidal anti-inlammatory drugs (NSAID) are effective in both cancer prevention and treatment of
established tumors6-8. Increasing evidence suggests that
the inlammation signiicantly contributes to the
etiol-ogy of EOC5,9. Collectively, hypotheses attributing the
EOC to ovulation, gonadotropin release, and hormonal inluences likely are not mutually exclusive as they all converge on the role of inlammation in promoting
ovarian tumorigenesis9,10. These indings result in the
initiation of a number of animal and clinical trials that examine the eficacy of cyclooxygenase (COX) inhibitors in primary and/or secondary prevention of cancer, both alone or as part of a combination therapy regimen for established tumors11.
To date, 48 ATP-binding cassette (ABC) transport-ers that are divided into seven families (ABC A-G) have
been identiied in the human genome12. Several of these
transporters can actively eflux a wide range of antican-cer drugs and reduce intracellular drug concentrations. This phenomenon eventually confers cross-resistance to various chemotherapeutics drugs, resulting in multidrug resistance (MDR) acquisition13,14.
The objective of this study was to determine the basic expression pattern of ABC transporters in an EOC cell line, and to investigate whether clinically relevant concentrations of acetaminophen and ibuprofen can inhibit the growth of EOC cells in vitro.
Methods
Cell culture
The human ovarian adenocarcinoma cell line TOV-21 G was obtained from the American Type Culture Collection (ATCC # CRL-11730), and spread according to their recom-mendations. These cells were maintained in a 1:1 mixture of culture medium 199 (Sigma # M2520) and MCDB 131 (Sigma # M8537), containing 10% fetal bovine serum (FBS)
at 37°C in a humidiied atmosphere of 5% CO2.
Cytotoxicity
Cell viability was determined through the use of 3-[4,5-dimethylthiazol-2-yl]-2,55-diphenyltetrazolium
bromide (MTT) colorimetric assay; in which MTT, a nontoxic pale yellow substrate, was converted to a dark blue formazan product by living cells. The formazan accumulation can be spectrophotometrically measured and it is directly proportional to the number of viable cells. All experiments were performed in quadruplicate.
Cells were plated at 1x105 cells/well in 96-well plates
and incubated at 37°C, for 24 hours. The cells were washed in the following day with PBS and treated with acetaminophen at concentrations ranging from 1.5 to 15 μg/mL, or ibuprofen at 2.0 to 20 μg/mL. Control wells contained no drug. After incubation periods of 24 and 48 hours, 10 μL of MTT tetrazolium solution was added to each well and the cells were incubated at 37ºC for three hours. Plates were then centrifuged and the supernatant was removed. The formazan was solubi-lized with 50 μL of DMSO, and absorbance at 550 nm was measured. The results of the treated cultures are expressed as the percentage of viable cells compared with untreated controls.
Fluorescence staining
Cell morphology was established after exposure to concentrations of 1.5, 7.5 and 15 µg/mL of acetamino-phen or 2, 10 and 20 µg/mL of ibuprofen for 48 hours through staining with DAPI, MitoTracker Orange and Live/Dead using a luorescence microscope (Axiovert 200, Zeiss).
ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually non-luorescent cell-permeant calcein AM to the intensely luorescent calcein. The polyanionic dye calcein is retained within live cells, producing an intense uniform green luores-cence in live cells. EthD-1 enters the cells with dam-aged membranes and undergoes a 40-fold enhancement of luorescence upon binding to nucleic acids, thereby producing a bright red luorescence in dead cells.
Real-time polymerase chain reaction
The quantitative polymerase chain reaction (PCR) was used to measure the expression level of human ABC transporter superfamily genes in TOV-21 G cells using a pre-made panel from Roche Applied Science. Each multi-well plate contains assays for 42 different ABC transporter genes in duplicate. Seven referential genes served as PCR controls and allowed for the quantiication of the relative expression of target genes. The assays were
carried out using a LightCycler® 480 instrument and 96
multi-well plates containing target-speciic primers and a FAM-labeled Universal Probe Library hydrolysis probe, which has RNA. Total RNA was isolated from TOV-21
G cells using TRIzol® reagent. Two micrograms of total
RNA were used to generate the irst strand cDNA through reverse transcription.
Statistical analysis
In order to compare the mRNA expression of dif-ferent ABC transporters, the results of gene expression were analyzed based on the cycle threshold (Ct) values normalized to beta-2-micro-globulin and 18 S prob-able dimethyladenosine transferase precursor genes. The Ct values of control genes were subtracted from those of the transporters to calculate a ΔCt value (e.g.,
Ct of transporter – CtB2M). The Student’s t-test was
performed to analyze the statistical signiicance of MTT values, and a one-way ANOVA was executed for pair-wise comparisons. Statistical analyses were done using GraphPad Software, Prism 4.0 (GraphPad Software, San Diego, CA, USA). Differences were considered statisti-cally signiicant if p<0.05.
Results
Cell proliferation
Acetaminophen at concentrations of 1.5, 7.5 and 15 µg/L decreased TOV-21 G cell proliferation in 3.5 (p=0.10), 3.5 (p=0.08), and 7.2% (p=0.02), respectively, at 24 hours compared to untreated control cells considered to be of 100% proliferation (Figure 1A). After 48 hours, acet-aminophen decreased cell proliferation in 7.6 (p=0.12), 5.2 (p=0.43), and 12.3% (p=0.01), as seen in Figure 1B.
When the cellular response was analyzed to 2, 10 and 20 µg/L of ibuprofen at 24 hours, decreases in cel-lular growth of 0.3 (p=0.96), 4.9 and 3.9% (p=0.05), respectively, were seen compared to controls (Figure 2A). These decreases were more apparent after 48 hours of ibuprofen exposure, at which time cellular growth was diminished to 7.8 (p=0.16), 9.52 (p=0.12), and 9.5% (p=0.04) compared to controls (Figure 2B).
Morphological changes
The morphological characteristics of cells were evalu-ated through luorescent staining. Forty-eight hours after treatment, cells that were treated with acetaminophen and ibuprofen did not show any morphological signs of apoptosis than did the controls (Figures 3 and 4A to D). The Live/Dead assay revealed that 48 hours of treatment
*
*
B A
Acetaminophen (µg/mL)
%Survival
1.5 0
25 50 75 100 125
7.5 15
Acetaminophen (µg/mL)
%Survival
1.5 0
25 50 75 100 125
7.5 15
Figure 1. Cell viability in ovarian cancer cell lines exposed to aceta-minophen in different time intervals. Bars represent the means of ex-periments performed in quadruplicate. Error bars indicate the standard deviation. Cell growth is expressed as a percentage of control. TOV-21 G cells showed a proliferation decrease at concentrations of 1.5, 7.5 and 15 µg/mL for drug exposure at (A) 24 and (B) 48 hours
with acetaminophen at 15 µg/mL or ibuprofen at 20 µg/mL did not increase the number of dead cells (Figures 3 and 4E to H).
MitoTracker Orange is an orange-luorescent dye that stains mitochondria in live cells. Cells were consid-ered MitoTracker Orange positive if a bright punctate orange luorescence of the mitochondria was observed, and negative if cells exhibited a diffuse orange cytoplasmic staining. By 48 hours, cells treated with acetaminophen or ibuprofen showed no morphological changes in their mitochondria (Figures 3 and 4I to L).
Real-time polymerase chain reaction
TOV-21 G cells had reduced expression of the ABCA1, ABCC3, ABCC4, ABCD3, ABCD4, and ABCE1 transporter
genes. The expression of ABCC4 (ΔCtB2M=-0.77 and ΔCt18S=-10.48) was not altered as much as that of ABCA1 (ΔCtB2M=-10.73 and ΔCt18S=-24,43), which was signiicantly under-expressed (Figure 5).
Discussion
Testing different drugs on cell lines is a critical component of the antineoplastic drug development. MTT assays allow for testing different concentrations and durations of drug exposure. Limited data are available regarding the effects of NSAIDS on cell lines of EOC15,16. The published literature includes a wide range of drugs, doses and schedules, making it challenging to reconcile the differences and to understand how well a drug may work in the clinical practice17-19.
From a clinically relevant perspective, ibuprofen is one of the most widely used analgesic, antipyretic and
anti-inlammatory drugs nowadays20. It has relatively
low risks for gastrointestinal, hepato-renal and other rare adverse drug reactions compared with other NSAIDs and
coxibs20. Indeed, it has been described as “the mildest
NSAID with the fewest side effects that has been in clinical
use for a long time”20. Patients have been maintained on
high doses of ibuprofen for years, without serious adverse
effects17. Advanced age has a minimal inluence on the
pharmacokinetics of ibuprofen, and dosage apparently
does not need to be adjusted for age21. The drug has,
in general, predictable and reliable kinetic properties20.
The maximum serum concentration (Cmax) following the
ingestion of 400, 600 and 800 mg doses is 15.4, 17.1
and 24.2 µg/mL, respectively20.
Acetaminophen is one of the most popular and widely used drugs for the treatment of pain and fever, and does not produce gastrointestinal damage or
un-toward cardiorenal effects22. Despite much research,
deinitive proof that the analgesic and antipyretic effects of acetaminophen are dependent on COX inhibition is still lacking22. Indeed, inhibition of a third form of COX, COX-3, is one of the more recent proposals that have been put forward to explain the unusual effects
of acetaminophen22,23. Plasma concentrations of 10 to
20 µg/mL have an assumed role to be therapeutic for
acetaminophen24.
To our knowledge, this is the irst report describ-ing the effect of ibuprofen and acetaminophen on the growth of an EOC cell line in vitro, using therapeutically relevant drug concentrations. Acetaminophen at a con-centration of 15 µg/mL showed a signiicant inhibition of an EOC cell line after 24 and 48 hours of treatment. Ibuprofen inhibited growth at 20 µg/mL, but only 48 hours after exposure. The anti-proliferative effect exerted by ibuprofen on the EOC cell line studied here
*
B A
Ibuprofen (µg/mL)
%Survival
2 0
25 50 75 100 125
10 20
Ibuprofen (µg/mL)
%Survival
2 0
25 50 75 100 125
10 20
Figure 2. Cell viability in ovarian cancer cell lines exposed to ibuprofen in different time intervals. Bars represent the means of experiments per-formed in quadruplicate. Error bars indicate the standard deviation. Cell growth is expressed as a percentage of control. TOV-21 G cells showed a proliferation decrease at concentrations of 2, 10 and 20 µg/mL for drug exposure at (A) 24 and (B) 48 hours
A E I
B F J
C G K
D H L
1.5 µg/mL
7.5 µg/mL
15 µg/mL Control
DAPI Live/Dead Mitotracker
Figure 3. Representative images of DAPI (A to D), Live/Dead (E to H) and MitoTracker (I to L) staining of TOV-21 G cells treated with 1.5, 7.5 and 15 µg/mL of acetaminophen. The white circles indicate red dead cells
is in accordance with previous observations1. The i nding that therapeutic levels of acetaminophen and ibuprofen have effects on EOC cells, suggests the possibility of a chemo-preventive/chemotherapeutic strategy using these agents in dosages and schedules that will minimize the side effects, while yielding optimal ovarian cancer prevention/treatment.
This study demonstrated that acetaminophen and ibuprofen at therapeutics levels do not activate apoptosis in EOC cells. The mechanism(s) underlying the decrease in cell proliferation remains to be elucidated. It has been
previously proposed that ibuprofen anti-proliferative actions are independent of COX and that p75NTR, a recently identii ed tumor suppressor, may in part be
responsible for ibuprofen anticancer properties1,25,26.
Previous studies suggest that acetaminophen is a tyrosine kinase substrate and intracellular glutathione depletion, reactive oxygen species formation and mitochondrial toxicity contributed to acetaminophen selective toxicity in melanoma cell lines27,28.
A E I
B F J
C G K
D H L
2 µg/mL
10 µg/mL
20 µg/mL Control
DAPI Live/Dead Mitotracker
Figure 4. Representative images of DAPI (A to D), Live/Dead (E to H) and MitoTracker (I to L) staining of TOV-21 G cells treated with 2, 10 and 20 µg/mL of ibuprofen. The white circles indicate red dead cells
Real-time PCR is the most reliable and sensitive gene expression proi ling technology for analyzing a panel of genes. Many studies have been carried out assessing the expression proi le of ABC transporters. Thus, most of these investigations analyze only one or a small group of ABC transporters. In contrast, our method allows for the complete analysis of 42 ABC transporters. In this study, we focused on determining the basic expression proi le of ABC transporters in TOV-21 G cells, an EOC cell line. Our data demonstrated that ABCA1, ABCC3, ABCC4, ABCD3, ABCD4 and ABCE1 were severely
∆Ct B2M ABCC4
0
-5
-10
-15
-20
-25
ABCD4 ABCE1 ABCC3 ABCD3 ABCA1
∆Ct B18S
Figure 5. Gene expression proile of different ABC transporters
normal-ized to a beta-2-micro-globulin precursor gene (ΔCt B2M=Cttransporter–
CtB2M) and an 18 S gene (ΔCt 18S=Cttransporter–Ct18S)
An increased understanding of the mechanisms underlying drug resistance may lead to the
develop-ment of more successful therapeutic protocols29. Hence,
the expression proile of ABC transporter genes in cell lines used for drug screening is highly important for the antineoplastic drug discovery.
These indings ensure further studies on the physi-ologic role, clinical relevance, and potential use of ABCC4 as a therapeutic target. Due to the limitations of in vitro
testing, these results should be conirmed in animal models for safety and eficacy. Using more than one cell type may help avoid making decisions based on tissue-speciic responses.
Finally, the therapeutic concentrations of acetamino-phen and ibuprofen directly inhibit EOC cell viability, which may signiicantly contribute to their antineoplastic effects. These results should encourage further research regarding the potential beneit of acetaminophen and NSAIDs in chemoprevention or as adjuvant therapies in EOC treatment.
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